Within the framework of the present Description, the term “parallel” is used with two different meanings depending on the context. Firstly, for example, the parallel determination of the individual sequence modules mentioned below is possible through the use of different primers, i.e. multiplex operation, and secondly, with specific reference to a method according to the invention, “parallel” amplification means the simultaneous amplification of nucleic acids both in the liquid phase and also in the solid phase by means of P3 primers described according to the invention.
The terms Probe, PCR Primer or Primer as the case may be will be used hereafter depending on the function of the oligonucleotide involved. An oligonucleotide that can be used as a primer for the specific amplification of target sequences can also be used as a probe to detect the target sequence.
A solid-phase microarray system and a method for the analysis of labelled (tagged) DNA sequences by hybridising onto probes immobilised onto a solid support and by determining the localisation of the labelling on the surface is known for example from EP 0 373 203.
A series of individual steps is necessary for a DNA microarray analysis of this kind. Thus the nucleic acid in the samples must be labelled directly or in the form of a copy, or alternatively the operation is performed with a collector nucleic acid on the microarray and a second detection probe in solution. At the same time, many applications such as the detection of biological contamination necessitate the amplification of the sample sequence, for example via a PCR. The (labelled) sample is then hybridised with the probe bonded onto the microarray in order to generate a specific, fixed-position signal which can be recorded and passed on, for example via a detection probe. This procedure gives rise to numerous individual reaction steps that make these analyses labour- and cost-intensive, slow and moreover error-prone.
The performing of a PCR in the presence of a solid support or chip (“PCR on Chip”) is in itself already known (Shoffner M. A., Cheng, J., Hvichia G. E., Kricka U., Wilding P.: Chip PCR. I. Surface passivation of microfabricated silicon-glass chips for PCR. Nucleic Acids Res. 1996 Jan. 15; 24(2): 375-9.; Cheng J., Shoffner M. A., Hvichia G. E., Kricka U., Wilding P.: Chip PCR. II. Investigation of different PCR amplification systems in microfabricated silicon-glass chips. Nucleic Acids Res. 1996 Jan. 15, 24(2): 380-5.
The principle of what is called “nested PCR” is also known (cf., for example, C. R. Newton, A. Graham: “PCR”, Spektrum Akademischer Verlag Heidelberg Berlin Oxford 1994, p. 59 and the other literature references cited there). Nested PCR involves the use of two PCR primer pairs, of which the members of one pair (known as the outer PCR primers) hybridise upstream and downstream respectively of the target DNA sequence that is to be amplified. The members of the second primer pair (known as the inner PCR primers) hybridise within the section that is pre-determined by the design of the first primer pair. The advantage of this technique lies in the considerable increase in sensitivity and specificity, because the target DNA sequence is amplified preferentially.
However, this technique is extraordinarily sensitive to contaminants of the kind that can be introduced, for example, even by pipetting together the reagents that are needed for the PCR. Moreover, in the case of a nested PCR it is necessary for an amplification that is separate in space and/or time to take place, i.e. for example that either the first amplicon must be diluted or at least one of the outer primers must be removed. Generally the dilution method is used, in which the concentration of the original matrix and the concentration of at least one of the outer primers (hereafter called P1 and P2 respectively) is greatly reduced. Although addition by pipette after the first amplification is possible in principle, it is associated with the risk that the advantages normally inherent in “nested PCR” (see above) will not be achievable. Furthermore, the person skilled in the art knows that when all three primers are present simultaneously in the mixture, no further increase in sensitivity and specificity is achievable, and for this reason it is no longer possible to call it a nested PCR in the real sense either (see for example WO 96/26291). For this reason WO 90/11369, WO 96/31622, WO 93/21339 and WO 98/28438 describe methods in which the amplification cycles take place separated in space and/or time.
Attempts were also made to pre-mix and appropriately stabilise the PCR reagents, particularly in order to simplify the conduct of the PCR reaction. However, there is no description in the state of the art to the effect that the reactants of the intended reaction are provided in dehydrated form directly on a microarray arrangement in such a way that the reaction can be started simply by adding the solution, preferably aqueous, of the sample.
Various different techniques for lyophilising or drying reagents to carry out nucleic acid analyses are described in the state of the art. Thus the preparation of hemispheres with all of the components necessary for a reaction, among them nucleic acids as well, is described in the U.S. Pat. No. 5,565,318. Similar techniques are used in the Applications FR 2 674 253, EP 0 298 669, EP 0 726 310, GB 2 333 358, WO 94/17106, WO 98/00530, U.S. Pat. No. 5,250,429 and EP 0 383 569.
All of the important nucleic acid techniques relating to the use of such reagent spheres are described in concrete terms in the Patent Application WO 96/32497. In addition, an apparatus or equipment in which the dried reagents for a sequencing reaction are used on a plate (presumably a micro-titration plate) is mentioned in the Patent Application WO 94/16107. A solid support in the form of a comb on which these reagents are present and which can be put onto what are known as “8's strips” or micro-titration plates in such a way that the reaction can proceed in the depressions of the micro-titration plates after the release of the reagents is described in Patent Application WO 96/17083.
The application of the individual PCR reagents in the form of spots spatially separated from one another (“Spots”) or even application over an area onto a solid phase in the form of a microscope slide is disclosed in Patent Application WO 93/14223. The reversible bonding of dried labelling (tagging) substances and the labelling of a probe after rehydration is also described. However, in this case the probe or probe nucleic acid itself is present expressly reversibly bonded to the solid support.
None of these techniques described in the state of the art enables parallel amplification in a coupled liquid/solid phase PCR system in which the desired results can be generated in a highly specific manner, with a high throughput rate and reproducibly—even in real time if desired—by evaluating an extension product that is formed detectably directly on the solid phase of the system and thus covalently bonded thereto, and can be passed on to the various areas of application.
The task of the invention is to eliminate the deficiencies present in the state of the art and to provide a favourably priced method for the amplification and analysis of DNA sequences that is simple and can be operated free from error, without needing to make any compromises regarding sensitivity and specificity.